A Empirical Study Of Treatment Mice Hepatic Carcinoma With M4-1BBL Gene Under The Control Of Mice Telomerase Reverse Transcriptase Gene Promotor

Object Construct a recombicant adenovirus vector which mice 4-1BBL gene (m4-1BBL) controled by mice telomerase reverse transcriptase promotor (mTERT promotor), compared with recombicant adenovirus vector m4-1BBL controled by CMV promotor, investigate the therapy effectiveness to mice hepatic carcinoma in vitro and in vivo.Mothods Clone mTERT promotor and m4-1BBL gene from C57BL/6 mice by PCR or RT-PCR respectively, verified it by direct sequence; sub clone the two genes into pAD/PL-DEST Gateway Vector according the protocol, construct the recombicant adenovirus pAD-mTERT-promotor-m4-1BBL and control plasmids pAD-CMV-m4-1BBL and pAD, Pacâ… linearizated them and transfected it into 293A cell by Lipofectamine2000, packaged and amplification them to: rAD, rAD-CMV-m4-1BBL and rAD-mTERT-promotor-m4-1BBL, cellular-immunochemistry confirm the virus amplificated successful. Transfected mice hepatic carcinoma cell strain Hepa1-6 and mice fibroblasts strain L929 with three recombicant adenoviruses, detected m4-1BBL expression on the two cell strains with m4-1BBL monoclonal antibodies by FCM; adding three viruses into two cells by MOI=10 density, MTT measure the influence of virus to growth of the two cells, detect apoptosis of the cells by Annexinâ…¤and PI; Extracted mice spleenic lymphocyte, purified it by Dynal negative selection immunomagnetic beads and activated mice spleenic T lymphocyte with PHA(phytohemagglutinin) or mice functional CD3, CD28 monoclonal antibodies respectively, adding the activated T lymphocyte into the two cells mentioned above which have been added three viruses, set up another group only adding activated T lymphocyte but adding no virus as another control group, MTT measure the influence of virus and activated T lymphocyte to growth of the two cells, detect apoptosis by Annexinâ…¤and PI, judge the inflence of adding virus and activated T lymphocyte to the two cells; Subcutaeous vaccinate different quantity Hepa1-6 cells to congenic mice, observe the tumor construction condition; Subcutaeous vaccinate 3×10⁶ quantity Hepa1-6 cells to congenic mice to construct mice hepatic carcinoma subcutaeous implantation model, intratumorally three virus to the mice hepatic carcinoma subcutaeous implantation model and observe the therapeutic action of the three virus, at the bobtail time, select tumor or tumor site tissues undertake pathology, select and draw-off the liver and blood examine at the same time; Transfected Hepa1-6 with the three virus, Subcutaeous vaccinate 3×10⁶ quantity transgenic Hepa1-6 cells to congenic mice to study the construct tumor ability of transgenic Hepa1-6 cell, select the tumor undertake pathology, observe the lymphocyte infiltrated condition.Result 1. Mice TERT promotor and mice 4-1BBL gene are coincidence with the sequence in GeneBank by direct sequence; 2. PCR verified that that the recombicant plasmids have or have no aiming gene; 3. Cellular immunochemistry verified that the virus packaged successful and the titer is 1×10¹⁰pfu/ml. 4. Transfect the three virus to Hepa1-6 and L929 with different titre, we cann't detect m4-1BBL at rAD group at any time and neither cell but detect m4-1BBL at rAD-CMV-m4-1BBL group at any time and two cell; we only detected m4-1BBL on Hepa1-6 cell and no on L929 cell at rAD-mTERT-m4-1BBL group; 5. Compare the three groups at different titre and different time, we find MOI=10 and 48 hours is the suitable titre and time for transfection. 6. Adding three virus to the two cell at MOI=10, we find that the three virus all have depressant effect on two cells, rAD-CMV-m4-1BBL group is the greatest group, there is significant difference among them with the other two virus(P<0.05); there is non-significant difference between rAD and rAD-mTERT-promotor-m4-1BBL(P>0.05); when detecting apoptosis, we find rAD-CMV-m4-1BBL has great promoting apoptosis effection on two cells, on the other hand, rAD-mTERT-promotor-m4-1BBL only has promoting apoptosis effection to Hepa1-6 and rAD has no effection to neither cell. 7. the purified mice spleenic T lymphocyte is found the expression of CD3 reaching 95%; activated the spleenic T lymphocyte by PHA or functional CD3CD28 respectively, we find that the cell gather under micro, detect CD69 and CD25 of the activated cell by FCM and found that the lymphocyte expression CD69 and CD25 reaching 50% at 48 hours; 8. Mixing the activated T cell with two cell which adding three virus respectively, MTT observe the growth of the two cells, we find that regardless of adding activated T lymphocyte only or adding each kind of virus, the growth is different with nomal cells(P<0.05); the group which adding activated T lymphocyte only has significant difference with other groups(P<0.05); the growth suppression is greater than adding rAD and rAD-mTERT-promotor-m4-1BBL group in mixing culture cell which adding rAD-CMV-m4-1BBL(P<0.05); there are non-significant difference between rAD and rAD-mTERT-promotor-m4-1BBL group(P>0.05); Detecting apoptosis of five group at 12, 24, 48 and 72 hours respectively, campared with adding virus simply, we find that mixing culture activated T lymphocyte with transfection cell can aggravate the apoptosis condition of two kinds of cells, especially rAD-CMV-m4-1BBL group; the apoptosis condition is obviously in T lymphocyte mixing culture Hepa1-6 cell group which adding rAD-mTERT-promotor-m4-1BBL and the condition is different in L929 cell; 9. Subcutaeous vaccinate 3×106 or 4×106 Hepa1-6 cells to congenic mice can successfully constructed tumor, it have significant difference among them with the other two quantity groups(1×106or 2×106)(P<0.05),but there is non-significant difference between3×106 and 4×106 group(P>0.05); 10. Subcutaeous vaccinate 3×106 Hepa1-6 cells to congenic mice to construct tumor, intratumorally the three virus into the tumor, we find when injecting rAD-CMV-m4-1BBL, the tumor deprivated almostly, and tumor obviously decreased size at rAD-mTERT-promotor-m4-1BBL group, there are no change at rAD group, the tumor of control group which injecting PBS only progressive enlargement; there are no mice death during experimental session; at bobtail time, we find ALT and AST in rAD-CMV-m4-1BBL group is higher that the other groups(P<0.05); there non-significant among the other groups(P>0.05); tissue slice show that there are no tumor cell on the tumor location at rAD-CMV-m4-1BBL group, only remain remnant (cell outline) of the tumor cell, has lymphnode hyperplasia, there are small pellet cancer cell nest in rAD-mTERT-promotor-m4-1BBL group, has lymphnode hyperplasia, necrosis focus scattered, cancer cell nest is small; the tumor cell in PBS group are different on size, alinement disturbance, karyomegaly, vacuole like, karyosome anachromasis, no necrosis or lymphocytes infiltration; the rAD group tumor cell are different on size, alinement disturbance, karyomegaly, vacuole like, karyosome anachromasis, has focus necrosis and lymphocytes infiltration, there are small amounts lymphocytes infiltration around zone of necrosis; there are anomalism hepatic plate and no lymphocyte infiltration in the rAD-mTERT-promotor-m4-1BBL and rAD-CMV-m4-1BBL group, the liver cell has light cytoplasm, good cell appearance, have no necrosis expression, there are no difference between two groups, there are no difference among the other groups with normal groups. 11. Post inoculation rAD-CMV-m4-1BBL transgenes tumor cell, there are no tumor growth, the tumor of vaccinated rAD-mTERT-promotor-m4-1BBL transgenes tumor cell has a small tumor and growth slowly, rAD and wild strain vaccination can construct a growth fast tumor, there are no-significant difference between them, the pathological section CD4 or CD8 detection find that rAD-mTERT-promotor-m4-1BBL transgenes cell has a obviously CD8 positive T lymphocytes infiltration and has significant difference with other groups(P<0.05), there are no-significant difference among other groups.Conclusion Recombicant adenovirus rAD-mTERT-promotor-m4-1BBL has the same effection with rAD-CMV-m4-1BBL and has target on tumor cell, campared with rAD-CMV-m4-1BBL, it is safer and fewer liver toxic and side-effect. So, rAD-mTERT-promotor-m4-1BBL is better than rAD-CMV-m4-1BBL.